Three-Component Reaction of 3-Formyl-6-Methylchromone, Primary Amines, and Secondary Phosphine Oxides: A Synthetic and Mechanistic Study

A fast, mild, and efficient catalyst-free approach has been developed for the synthesis of chromonyl-substituted α-aminophosphine oxides by the three-component reaction of 3-formyl-6-methylchromone, primary amines, and secondary phosphine oxides at ambient temperature. Carrying out the reaction with aliphatic amines or aminoalcohols at a higher temperature (80 °C), phosphinoyl-functionalized 3-aminomethylene chromanones were formed instead of the corresponding chromonyl-substituted α-aminophosphine oxides. No reaction occurred when 3-formyl-6-methylchromone and secondary phosphine oxides were reacted with aromatic amines in the absence of any catalyst. Applying a basic catalyst, the formation of the phosphinoyl-functionalized 3-aminomethylene chromanones was observed; however, the reaction was not complete. Detailed experimental and quantum chemical studies were performed to study the transformation. Moreover, the in vitro cytotoxicity of phosphinoyl-functionalized 3-aminomethylene chromanones was also investigated in three different cell lines, such as human lung adenocarcinoma (A549), mouse fibroblast (NIH/3T3), and human promyelocytic leukemia (HL60) cells. Several derivatives showed modest activity against the human promyelocytic leukemia (HL60) cell line.


INTRODUCTION
In the last few decades, multicomponent reactions (MCRs) became widely applied in the field of small-molecule drug discovery. 1 Starting from simple and cheap reagents, a large library of structurally related compounds can be synthesized in a short time. 2 In general, MCRs have high atom efficiency, which saves time and energy. 3 One of the most important groups of organic compounds is heterocyclic derivatives. They play an important role in the pharmaceutical and plastic industries and in agriculture. 4 Among heterocyclic compounds, O-heterocycles are found in the structure of naturally occurring vitamins, hormones, antibiotics, sugars, and pigments. 5 The synthetic derivatives can also have great pharmaceutical significance, as they have antitumor, antiviral, antimicrobial, or anti-inflammatory activities. 6 In addition, some of them may also have photochemical properties. 7 3-Formylchromone, as an interesting starting material in MCRs, has three different electron-deficient centers that can be attacked by various nucleophiles, thereby providing an opportunity to create a wide range of new chromone derivatives. 8 In addition, intramolecular ring opening can also take place, and further heterocycles can be formed. 9 Several derivatives containing a chromone backbone have already been synthesized; however, only a few chromones containing a phosphine oxide or a phosphonate moiety can be found in the literature. Some of them have been applied as flame-retardant agents in polymers 10 or have antioxidant, antimicrobial, 11 or anticancer effects (against antitumor HepG2 (human liver) and HFB4 (human normal melanocyte) cell lines). 12 A few chromonyl α-aminophosphonates were prepared by the three-component Kabachnik−Fields reaction of 3formylchromones, aromatic amines, and dialkyl phosphites. 11,13 The condensations were carried out without a catalyst in a solvent or in the absence of any solvent, at 70−110°C for long reaction times (2−6 h), and the desired chromonyl α-aminophosphonates were obtained in variable yields (16− 70%). In another example, 3-formylchromone derivatives, 3amino-2-phenyl-quinazolin-4(3H)-one, and 1.5 equivalents of diethyl phosphite were reacted in a "one-pot" method or in two steps, where a Schiff base was formed first and then it reacted with the phosphorus reagent. 12 Additional aminophosphonates were synthesized by the Kabachnik−Fields reaction of 3-formylchromones, amide derivatives, and trialkyl phosphites at 80−100°C for 1−1.5 h in acetic acid as the solvent. 14,15 In one case, the condensation of 3-formylchromone, bifunctional amines (aminoalcohols or diamines) or urea derivatives, and dialkyl phosphites was investigated, where a ring opening was observed, and new five-, six-or sevenmembered heterocyclic units were formed. 16 In this paper, the three-component reaction of 3-formyl-6methylchromone, primary amines, and secondary phosphine oxides was investigated in the absence of any catalyst or using a basic catalyst. We aimed at the optimization of a model reaction, and to extend it using various amines and phosphorus reagents. We also wished to study the mechanism of the threecomponent reaction by experiments, as well as by quantum chemical calculations. The in vitro cytotoxicity of several compounds synthesized was also aimed to be investigated in three different cell lines (human lung adenocarcinoma cell line (A549), mouse fibroblast healthy cell line (NIH/3T3), and human promyelocytic leukemia cell line (HL60)).

Three-Component Reaction of 3-Formyl-6-methylchromone, Primary Amines, and Secondary
Phosphine Oxides. The catalyst-free Kabachnik−Fields reaction of 3-formyl-6-methylchromone, butylamine and diphenylphosphine oxide (DPPO) was studied with respect to the heating mode, temperature, and reaction time (Table 1). First, the condensation was carried out in acetonitrile, at 80°C for 1 h in an oil bath. The conversion was not complete, and surprisingly beside the corresponding chromonyl-substituted α-aminophosphine oxide (1a), a phosphinoyl-functionalized 3-(butylamino)methylene chromanone (2a) was formed as the major component (Table 1, entry 1). When repeating the reaction under microwave (MW) heating, the conversion was complete; however, the proportion did not change (Table 1, entry 2). After column chromatography, the phosphinoylfunctionalized 3-(butylamino)methylene chromanone (2a) was obtained in a yield of 86%. In order to increase the ratio of the α-aminophosphine oxide product (1a), the temperature was decreased to 60°C, and it was found that the ratio of products 1a and 2a was reversed (64:36) ( Table 1, entry 3). When carrying out the reaction at room temperature for 1 h without MW irradiation, the ratio of product 1a increased to 76%, which did not change further after longer reaction time (4 h) ( Table 1, entries 4 and 5) or at lower temperature (0°C ), where the conversion was lower, but the product composition was similar (Table 1, entry 6). After 4 h reaction time at ambient temperature and column chromatography, 1a was isolated in a yield of 49%. By slow evaporation of the acetonitrile solution of compound 1a, single crystals suitable for the X-ray diffraction (XRD) study were obtained, which revealed the molecular structure of compound 1a (Figure 1).
In the crystal lattice, the amino group of 1a molecules is involved in the formation of hydrogen bonded dimer units via centrosymmetric N−H···O�P interaction ( Figure S1, Table  S1 in the Supporting Information). These dimers are further connected into a chain along the c axis through π···π interactions between two adjacent chromenyl C4−C9 rings with a centroid-to-centroid distance of 3.683 Å ( Figure S2).
After that, the catalyst-free reaction of 3-formyl-6-methylchromone, aniline, and diphenylphosphine oxide was studied (Table 1, entries 7−9). Performing the condensation at room temperature for 30 min without MW irradiation, the conversion was already 82%, and the chromonyl-substituted  α-aminophosphine oxide (1b) was formed selectively (Table 1,  entry 7). Increasing the reaction time to 1 h, no diphenylphosphine oxide remained in the mixture, and product 1b was isolated in a yield of 94% (Table 1, entry 8). In order to investigate the formation of the phosphinoyl-functionalized 3-(phenylamino)methylene chromanone (2b), the reaction was also carried out at 80°C for 1 h in an MW reactor; however, no product 2b was detected even at a higher temperature of 120°C (Table 1, entries 9 and 10).
In the next part, the reaction of 3-formyl-6-methylchromone, aniline, and diphenylphosphine oxide was carried out in the presence of basic catalysts to investigate the formation of the phosphinoyl-functionalized 3-(phenylamino)methylene chromanone (2b) ( Table 2). When using 10 mol % of pentamethyldiethylenetriamine (PMDTA) as the catalyst and performing the reaction in acetonitrile at 80°C for 1 h in an MW reactor, the conversion was almost complete (98%), and the mixture mainly contained chromonyl-substituted αhydroxyphosphine oxide (3) (50%); however, besides the chromonyl-substituted α-aminophosphine oxide (1b), 28% of 3-(phenylamino)methylene chromanone derivative (2b) was also formed (Table 2, entry 1). By increasing the amount of PMDTA to 20 mol %, the ratio of products 1b and 2b increased to 24 and 34%, respectively (Table 2, entry 2). When switching to Hunig's base (N,N-diisopropylethylamine, DIPEA) and repeating the reaction at 80°C for 1 h, the conversion was 90%; however, the ratio of compounds 1b, 2b, and 3 changed to 47:30:13, which means that in this case, the formation of the α-aminophosphine oxide derivative (1b) was more favorable (Table 2, entry 3). When the condensation was performed in the presence of 20 mol % of DIPEA at a higher temperature (100°C) and for a longer reaction time (2 h), a complete conversion was achieved, and the phosphinoylfunctionalized 3-(phenylamino)methylene chromanone (2b) was the main component (54%) (Table 2, entry 4). One can conclude that when using aniline in the three-component reaction, (Z)-3-[(amino)methylene]-2-(diphenylphosphoryl)-6-methylchroman-4-one (2b) can only be synthesized under harsher conditions, in the presence of a basic catalyst at a higher temperature with a longer reaction time, however, not selectively.
During the investigation of the mechanism of the formation of products 1a/1b and 2a/2b, it could be established that two different reaction paths (Path I and Path II) could be considered, as depicted in Scheme 1.
In order to get a deeper insight into the mechanism (Scheme 1), different intermediates involved in the postulated reaction paths were synthesized. Moreover, density functional theory (DFT) calculations were performed to support our findings and help to understand the reaction mechanism at the ωB97X-D/6-31G* level (more details and comparison of different levels of theory in the SI).
First, the thermodynamic stability of compounds 1a, 1b and 2a, 2b was calculated. According to our DFT calculations at the ωB97X-D/6-31G* level, the energy difference between the two isomers is tiny. 2a is more stable by 1.0 kcal/mol than 1a; in contrast, 2b is less stable by 1.1 kcal/mol than 1b. Application of different levels of theory does not give significant differences in energy (Table S2). It is important to mention that these energy differences are small (they are actually below the accuracy of the theoretical level); therefore, no significant thermodynamic control could be expected. The direct (monomolecular) transformation of 1a to 2a and that of 1b to 2b were calculated, and as it can be expected, monomolecular barriers are extremely high (120.0 and 80.1 kcal/mol, respectively). It is in agreement with the high stability of the isolated solid compound (1b), which was heated until 120°C, and no decomposition or change was observed based on 31 P NMR signals.
After the evaluation of the thermodynamic situation and exclusion of the direct transformation of 1a/2a to 1b/2b, the reaction of 3-formyl-6-methylchromone with diphenylphosphine oxide was carried out (Scheme 1/Path I). Complete conversion was achieved in acetonitrile at room temperature after 1 h, and the corresponding chromonyl-substituted αhydroxyphosphine oxide (3) was formed in a yield of 98%. After that, derivative 3, which can be considered as an intermediate of the three-component reaction, was reacted further with aniline at room temperature for 1 h. This reaction was complete, and only the desired chromonyl-substituted αaminophosphine oxide (1b) was formed. A similar reaction with butylamine (at 25°C for 1 h) gives compound 1a as the main product; however, the conversion was only 58%, which did not change significantly when the reaction was allowed to stir for a longer time. When repeating this reaction at a higher temperature (80°C for 1 h), the 2-phosphinoyl-and 3-(butylamino)methylene-substituted 6-methylchroman-4-one derivative (2a) was obtained with a conversion of 93%, while a similar reaction with aniline did not give the corresponding 2b product. One noticeable difference between the two reactions sets is the amine's basicity, which seems crucial toward the products 2a and 2b. Our DFT calculations further supported this finding, and the postulated mechanism of the formation of 2a and 2b is shown in Scheme 2. The nucleophilic attack of the amine and the ring opening step has a high barrier in both cases (61.3 and 73.9 kcal/mol, respectively), and in the transition states, the H changes its position from the amine nitrogen toward the ring's oxygen. Usually, the transition state of proton migration could be stabilized by polar solvents and by the presence of a coordinative water molecule (it is a byproduct of the reaction); therefore, a lower barrier could be expected for the real system, where solvent-assisted proton transport may significantly reduce this barrier (which was calculated without coordinative water/solvent molecules in a recent study).
The following step is the nucleophilic attack of the phenolic −OH group. All attempts to localize the corresponding transition states failed in our hand, and further scan calculations showed that the reaction could not proceed this way. During these scan calculations, the distance between the phenolic oxygen atom and the α-carbon atom was gradually decreased by 0.1 Å, at each bond length the geometry of the structure was optimized, and the energy of the system was investigated. The energy of the system increased strictly monotonic, without any local maxima, which indicates that no local minima could be expected for an intermediate, in which the phenolic oxygen was bonded to the α-carbon atom. On the other hand, after deprotonation of the acidic phenolic −OH group, and calculating the system as an anion, the attack of the −O − to the α-carbon atom is barrierless. This simple model clearly indicates the crucial role of the basic conditions. While aliphatic amines, which are more basic than aniline (ΔpK a ∼ 3), provide sufficiently basic conditions, the use of less basic aniline in the reaction without any additional base renders the ring closing step impossible. In the presence of a catalytic amount of external base, the reaction proceeds smoothly due to lower energy barriers of the pathway, including deprotonation−protonation steps.
In the next part, we have investigated the second possible reaction route (Scheme 1/Path II). The condensation of 3formyl-6-methylchromone and butylamine or aniline was carried out at room temperature. Schiff base 10 (R = Bu) was formed in a conversion of 74%, and Schiff base 11 (R = Ph) was obtained with a somewhat higher conversion (78%). Then, the reaction mixtures containing imine 10 or 11 were reacted further with diphenylphosphine oxide at room temperature. In both cases, the reactions were incomplete after 1 h. When starting from imine 11 only the corresponding α-aminophosphine oxide derivative (1b) was formed with 76% conversion; however, in case of Schiff base 10 (R = Bu) the conversion was lower (50%), and besides α-aminophosphine oxide 1a (32%), the phosphinoyl-functionalized 3-(butylamino)methylene chromanone (2a) was also formed (18%). Because of the low conversion, the reaction of imine 10 and diphenylphosphine oxide was repeated at 80°C (with 1 h reaction time). In this case, the 3-(butylamino)methylene chromanone derivative (2a) was formed as the major product (66% conversion). In view of these data, this reaction route (Path II, starting from the corresponding Schiff bases 10 or 11) seems to be much slower than Path I. One possible explanation could be the significantly higher barrier of the reaction between the corresponding Schiff base and of diphenylphosphine oxide (compared to the barriers in Path I). Despite the fact that several direct additions of diphenylphosphine oxide to multiple bonds were reported, 17 these reactions require harsher reaction conditions (high temperature or strong bases (such as KOH and t-BuOK)). We were not able to localize any transition state between the corresponding Schiff bases (10 and 11) and diphenylphos-Scheme 2. Mechanism of the Formation of (Z)-3-[(Butylamino)methylene]-2-(diphenylphosphoryl)-6-methylchroman-4-one (2a) phine oxide toward compounds 1a, 2a and 1b, 2b. On the one hand, the reaction could proceed through the tautomer form of diphenylphosphine oxide (diphenylphosphinous acid). 18 The nucleophilic attack and the migration of the proton have medium high barriers 10.2−27.4 kcal/mol (Scheme 3). On the other hand, it is important to highlight that the presence of the less stable tautomer form of diphenylphosphine oxide (even if it is stabilized by the H-bond with the amine base) is required for this reaction route. Finally, the model system was investigated in which diphenylphosphine oxide was deprotonated. In this particular case, the addition is barrierless, in agreement with the former finding in case of harsher reaction conditions. 17 Alternatively, the corresponding Schiff bases (10 and 11) are able to hydrolyze to the corresponding starting compounds (aldehyde and amine), and the reaction could proceed through Path I as well. In view of our experimental and theoretical results, both reaction paths are possible.
A similar reaction with α-aminophosphine oxide 1b was also performed in acetonitrile under the same conditions. However, the formation of the phosphinoyl-functionalized 3-(phenylamino)methylene chromanone (2b) was not observed at all (Scheme 5). Carrying out an experiment in the presence of 10 mol % of DIPEA, α-hydroxyphosphine oxide (3) and the enamine-type derivative (2b) were formed from the αaminophosphine oxide (1b). This also confirms that the formation of the phosphinoyl-functionalized 3-(phenylamino)methylene chromanone (2b) is not possible without a basic catalyst, if the nitrogen atom is substituted with an aromatic moiety. Furthermore, the low conversion was in agreement with the somewhat lower (1.1 kcal/mol) stability of compound 2b compared to 1b.
After optimization and understanding the reaction mechanism, our aim was to synthesize a small library of structurally related new derivatives by the extension of the model reaction (Scheme 6). First, the Kabachnik−Fields reaction of 3-formyl-6-methylchromone, aromatic amines, and secondary phosphine oxides was performed under the optimized conditions (at 25°C for 1 h, without a catalyst, in acetonitrile) found earlier ( Table 1, entry 7). The condensation of 3-formyl-6methylchromone, aniline, p-anisidine or 4-chloroaniline, and diphenyl phosphine oxide resulted in the corresponding 3- Neither the electron-donating methoxy group nor the electronwithdrawing chloro-substituent caused a significant difference in selectivity, leading to the formation of the α-aminophosphine-oxide derivatives (1c and 1d) as the major products. 3-Formyl-6-methylchromone and aniline were also reacted with bis(p-tolyl)-, bis(3,5-dimethylphenyl)-or with bis(2-naphthyl)phosphine oxide, where further three new chromonyl-substituted α-aminophosphine-oxides were synthesized (4, 5, and 6) selectively in yields of 93−95%.

CONCLUSIONS
In summary, we have developed a novel and practical catalystfree method for the synthesis of new chromonyl-substituted αaminophosphine oxides (1a−d and 4−6) by the Kabachnik− Fields reaction of 6-methyl-3-formylchromone, secondary phosphine oxides, and primary amines at ambient temperature with a short reaction time. This procedure means a promising approach to attain these new heterocycles, since it applies mild and easily operational conditions (no special reagents, catalysts or additives, no heating). In addition, we have shown that by carrying out the catalyst-free three-component reaction with aliphatic amines or aminoalcohols at higher temperature (80°C ) under MW irradiation, enamine-type derivatives (2a−g and 7a−c, 8a−c and 9a−c) were formed instead of chromonyl-substituted α-aminophosphine oxides (1a−d and 4−6). The methodology was applied for the synthesis of a wide range of phosphinoyl-functionalized 3-(amino)methylene chromanones (2a−g and 7a−c, 8a−c and 9a−c), which form a new family of compounds in the literature. In case of aromatic amines, the enamine-type derivatives could be only prepared in the presence of a base; however, this reaction was not complete. Detailed experimental and quantum chemical studies have revealed that the phosphinoyl-functionalized 3-(amino)methylene chromanone derivatives could be formed by a ring opening of the chromone ring. This transformation depends on the basicity of the amines used in the synthesis; therefore, it can easily take place with aliphatic amines. In case of aromatic amines, an additional base had to be used.

ACS Omega http://pubs.acs.org/journal/acsodf Article
The crystal structure of compound 1a was also studied by single-crystal XRD analysis. Furthermore, an intermediate (3) supporting the mechanism of the reaction was also identified and isolated.

General Information.
All starting materials were purchased from commercial sources and were used without further purification. The MW-assisted reactions were carried out in a 300 W CEM Discover focused MW reactor (CEM Microwave Technology Ltd., Buckingham, UK) equipped with a pressure controller using 10−20 W irradiation under isothermal conditions. The reactions under conventional heating were carried out in an oil bath.
High-performance liquid chromatography-mass spectrometry measurements were performed with an Agilent 1200 liquid chromatography system coupled with a 6130 quadrupole mass spectrometer equipped with an ESI ion source (Agilent Technologies, Palo Alto, CA, USA). Analysis was performed at 40°C on a Gemini C18 column (150 mm × 4.6 mm, 3 μm; Phenomenex, Torrance, CA, USA) with a mobile phase flow rate of 0.6 mL/min. The composition of eluent A was 0.1% (NH 4 )(HCOO) in water; eluent B was 0.1% (NH 4 )(HCOO) and 8% water in acetonitrile, 0−3 min 5% B, 3−13 min gradient, 13−20 min 100% B. The injection volume was 2 μL. The chromatographic profile was registered at 254 nm. The MSD operating parameters were as follows: positive ionization mode, scan spectra from m/z 120 to 1000, drying gas temperature 300°C, nitrogen flow rate 12 L/min, nebulizer pressure 60 psi, and capillary voltage 4000 V.
High-resolution mass spectrometric measurements were performed using a Sciex 5600+ Q-TOF mass spectrometer in positive electrospray mode.
The 1 H, 13 C, and 31 P NMR spectra were taken in CDCl 3 solution on a Bruker AV-300 spectrometer operating at 300, 75.5, and 121.5 MHz, respectively. The chemical shifts (δ) are reported in parts per million (ppm) and downfield relative to 85% H 3 PO 4 , as well as TMS, the coupling constants (J) are reported in Hz. (1a−d, 4−6). A mixture of 1.0 mmol of 6-methyl-3-formylchromone (0.12 g), 1.0 mmol of secondary phosphine oxide (0.20 g of diphenylphosphine oxide, 0.23 g of bis(p-tolyl)phosphine oxide, 0.26 g of bis(3,5dimethylphenyl)phosphine oxide or 0.30 g di(naphthalene-2yl)phosphine oxide), and 1.0 mmol of amine (0.10 mL of butylamine, 0.09 mL of aniline, 0.12 g of p-anisidine or 0.13 g of 4-chloroaniline) was stirred in 1 mL of acetonitrile at 25°C for 1−4 h. The products were purified by column chromatography using silica gel as the solid phase and dichloromethane-methanol (97:3) as the eluent. The following products were thus prepared.